Lutein, lutein composition and application thereof

Abstract

The present invention discloses lutein, a lutein composition and its application, and belongs to the field of biomedicine technology. The preparation method of lutein comprises the following steps: extracting marigold flower particles with propane and butane as extraction solvents, then distilling and concentrating the obtained extract after two evaporations, and then removing the solvent from the obtained extract and centrifuging it to obtain the lutein. The present invention also discloses a lutein composition, comprising the following raw materials: lutein, zeaxanthin, curcumin, quercetin, fucoidan, honeysuckle extract, casein, beeswax, soybean lecithin, soybean oil, glycerin and hydroxypropyl methylcellulose. The raw materials of the lutein composition of the present invention are natural, all of which are food-grade or medicinal and edible ingredients, have no side effects, have low production and manufacturing costs, have good consumer or patient compliance, can be used for a long time, and have a dual effect, thereby achieving the effect of conditioning and improving body inflammation to prevent and improve IBD and thrombotic complications.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to lutein, a lutein composition and applications thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory disease characterized by unknown etiology, chronic inflammation, and recurrent episodes. It includes two main disease types: Crohn's disease (CD) and ulcerative colitis (UC). It is worth noting that extraintestinal complications of IBD are one of the important reasons for the recurrent attacks and high mortality of patients.

[0003] In recent years, thromboembolism (TE) has garnered increasing attention as an extraintestinal complication of IBD, often occurring during flares of IBD disease activity. Thromboembolism, which primarily includes atherosclerosis, varicose veins, atrial fibrillation, pulmonary embolism, and acute myocardial infarction, is a serious and potentially life-threatening event. For many years, the risk of TE in patients with IBD has been increased, with the overall incidence of systemic TE events reported to be 6.2% in patients with IBD, a threefold increase compared to the general population. Evidence suggests that inflammation and coagulation are interdependent physiological processes, with inflammatory responses promoting coagulation and vice versa, potentially triggering a vicious cycle. IBD is associated with a hypercoagulable state and an increased risk of thromboembolism, with evidence suggesting that thrombi can form in both inflamed intestinal and extraintestinal tissues.

[0004] The hypercoagulable state and enhanced thrombosis observed in human IBD have been reproduced in animal models of colitis. Animal studies have also shown that increased levels of procoagulants (such as thrombin) and decreased levels of endogenous anticoagulants (such as activated protein C) contribute to the propagation of intestinal inflammation in experimental IBD. There is also evidence that the expression of tissue factor and related proteins changes during the development of experimental colitis-related thrombosis. IBD combined with venous thrombosis is closely related to abnormal activation of the procoagulant system, including activation of the coagulation system, downregulation of natural anticoagulant mechanisms, impaired fibrinolysis, increased platelet counts, and endothelial cell dysfunction. Relevant data indicate that the hypercoagulable state of blood in IBD is not caused by a specific mechanism, but rather by the interaction of a very large system.

[0005] The treatment and prevention of venous thrombosis in patients with IBD are crucial for the effectiveness of IBD treatment. Chinese guidelines suggest that prophylactic anticoagulation therapy primarily includes pharmacologic anticoagulation and mechanical prophylaxis. Anticoagulants primarily include low molecular weight heparin, pentosan heparin, and / or fondaparinux sodium. Mechanical prophylaxis primarily utilizes graduated compression stockings (GCS), intermittent pneumatic pressure devices (IPS), and plantar pressure pumps (VFPs). However, pharmacologic treatments can be associated with adverse reactions, such as skin bleeding, intraperitoneal bleeding, hematuria, fever, headache, chills, nausea, vomiting, constipation, epistaxis, ecchymosis, mild hematuria, and allergic reactions. Furthermore, drug interactions must be carefully considered, leading to poor patient compliance. Compared with anticoagulant therapy, mechanical prophylaxis is more acceptable to patients and simpler to implement. However, it is not suitable for patients with severe lower limb edema, pulmonary edema, or dermatitis. Its application is limited, its cost is high, and it even carries the risk of serious infection and malignancy. Therefore, there is an urgent need to find an effective and inexpensive drug or method that can effectively relieve the symptoms of IBD and reduce thrombosis.

[0006] Lutein is a xanthophyllic carotenoid found in various foods, such as dark green leafy vegetables and egg yolks. Lutein exhibits antioxidant activity and scavenges reactive oxygen species, such as singlet oxygen and lipid peroxyl radicals. Oxidative stress activates inflammatory mediators, leading to the development of metabolic and inflammatory diseases. Recent basic and clinical studies have investigated the anti-inflammatory effects of lutein based on its antioxidant activity and modulation of oxidant-sensitive inflammatory signaling pathways. Therefore, it may protect against various inflammatory diseases, including neurodegenerative diseases, diabetic retinopathy, osteoporosis, cardiovascular disease, skin diseases, liver damage, obesity, and colitis. Furthermore, lutein has tissue-specific effects, such as regulating lipid profiles in cardiovascular disease, adipocyte differentiation in obesity, and skin function. Therefore, consuming foods rich in lutein may help prevent inflammatory diseases caused by oxidative stress.

[0007] Therefore, there is an urgent need to find an intervention program developed using lutein that has high universality, good patient compliance, low development cost, no side effects, and can simultaneously prevent and improve IBD and thrombotic complications, in order to prevent and improve IBD and thrombotic complications, improve the quality of life of IBD patients, and provide reference for the clinical development of related drugs. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a lutein composition and a preparation method and application thereof.

[0009] To achieve the above objectives, the present invention provides the following technical solutions:

[0010] A method for preparing lutein comprises the following steps:

[0011] The marigold flower particles are extracted using propane and butane as extraction solvents, and the obtained extract is evaporated twice and then distilled and concentrated. The solvent is removed from the obtained extract and then centrifuged to obtain the lutein.

[0012] Preferably, in the extraction solvent, the volume ratio of propane to butane is 1:(1.1-1.4).

[0013] Preferably, the mass ratio of the extraction solvent to the marigold flower particles is 1:(2-3).

[0014] Preferably, the extraction temperature is 40° C., the pressure is 0.8-0.9 MPa, and the time is 45-60 min.

[0015] Preferably, the evaporation temperature is 36°C ± 2°C and the pressure is 0.04-0.05 MPa;

[0016] The temperature of the distillation concentration is 60° C. and the pressure is 0.04-0.05 MPa.

[0017] Beneficial Effects: The lutein in the present invention is a natural extract. Lutein is a heat-sensitive substance, sensitive to light and heat, and easily oxidized and deteriorated. Its physical and chemical properties dictate that the production process must be carried out in a closed environment at room temperature, so the entire extraction process is carried out within a pressure vessel. The present invention utilizes low-temperature extraction technology from fresh marigold flowers. Under specific temperature, pressure, and solvent conditions, the extraction rate and purity of natural lutein are improved, thereby increasing the purity of the lutein in the composition.

[0018] A method for preparing lutein provides lutein prepared by the method.

[0019] The invention discloses an application of lutein in the preparation of a medicine for preventing and improving IBD and its thrombotic complications.

[0020] A lutein composition comprising the following raw materials in different weight fractions:

[0021] The above-mentioned lutein 8-13.5%, zeaxanthin 5-12%, curcumin 8-16%, quercetin 3-21%, fucoidan 10-13%, honeysuckle extract 6-11.3%, casein 0.8-1.5%, beeswax 0.4-1.2%, soybean lecithin 0.4-1%, soybean oil 5.5-24.2%, glycerin 8-19% and hydroxypropyl methylcellulose 5-7.5%.

[0022] Beneficial Effects: The fucoidan in this invention forms a complex with lutein, changing its physical form from a poorly soluble crystalline form to a more soluble amorphous state. This transformation increases lutein's solubility in the gastrointestinal tract, thereby improving its absorption efficiency. Furthermore, the fucoidan-lutein complex is unstable due to weak interfacial interactions, resulting in a supersaturated state in aqueous solution, effectively inhibiting lutein precipitation and increasing its solubility. Quercetin interacts with casein, forming a less hydrophobic complex through covalent or non-covalent interactions, which acts as an encapsulation and transport agent for lutein, enhancing its bioaccessibility. Hydroxypropyl methylcellulose forms a stable supersaturated system through multiple hydrogen bonds, promoting lutein's absorption and utilization. Glycerin acts as a binder and stabilizer in the preparation of lutein preparations. Curcumin and honeysuckle extracts are effective in improving intestinal inflammation and thrombosis, synergizing with lutein and zeaxanthin to alleviate symptoms of IBD and extraintestinal thrombosis.

[0023] Preferably, the dosage form of the lutein composition includes one or more of powder, emulsion, capsule and soft candy dosage forms.

[0024] Beneficial effects: Based on the mechanism of mutual influence between IBD and thrombosis, the present invention screens and optimizes a lutein composition that can effectively prevent and improve IBD and thrombotic complications, and explores the mechanism of action and possible pharmacological signaling pathways of the optimal formula lutein composition for improving IBD and thrombotic complications. It is confirmed that the lutein composition of the present invention can effectively improve IBD and thrombotic complications by regulating inflammatory factors IL-6, IL-1β, TNF-α and thrombus flow time. The lutein composition of the present invention is made of natural raw materials, all of which are food-grade or medicinal and edible ingredients, have no side effects, have low production costs, have good consumer or patient compliance, can be used for a long time, and have a dual effect, thereby achieving the effect of regulating and improving body inflammation to prevent and improve IBD and thrombotic complications.

[0025] A lutein composition is used in the preparation of a medicine for preventing and improving IBD and its thrombotic complications.

[0026] Beneficial effects: The lutein composition provided by the present invention has an improving effect on intestinal inflammation and extraintestinal thrombosis. It is because it regulates inflammatory factors in the inflammation-coagulation related signaling pathway, inhibits the expression of pro-inflammatory factors, reduces the inflammatory response and changes in the coagulation mechanism, and improves the flow of thrombus, thereby playing a good regulatory role in colitis and related extraintestinal thrombosis.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] The present invention provides a lutein composition that can effectively prevent IBD and its intra- and extra-intestinal thrombotic complications, as well as its preparation method and application. It is the first time that it is discovered that a lutein composition can simultaneously prevent and improve inflammatory bowel disease and corresponding thrombotic complications. The lutein of the present invention is obtained by preparing fresh marigold flowers through solvent subcritical low-temperature extraction technology. The present invention further confirms through animal experiments that the composition of the present invention can effectively regulate the symptoms of colitis and extra-intestinal thrombotic complications by regulating inflammatory factors and thrombotic changes in the inflammation-coagulation system. It has great clinical development and application value and provides great reference significance for the clinical development of inflammation-coagulation drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0030] Figure 1 The results are for weight loss of mice in the experimental group;

[0031] Figure 2 The diarrhea results of mice in the experimental group;

[0032] Figure 3 The results of bloody stools in the experimental group mice;

[0033] Figure 4 The macroscopic scores of mice in the experimental group are shown in Figure 2.

[0034] Figure 5 This is the result of vascular occlusion time of mice in the experimental group;

[0035] Figure 6 The results of peripheral blood cell analysis (neutrophils) of mice in the experimental group;

[0036] Figure 7 The results of peripheral blood cell analysis (platelets) of mice in the experimental group;

[0037] Figure 8 The thrombus weight results of mice in the experimental group;

[0038] Figure 9 The thrombus length results of mice in the experimental group;

[0039] Figure 10 This is the tail bleeding time result of mice in the experimental group;

[0040] Figure 11 The results of hemoglobin levels of mice in the experimental group;

[0041] Figure 12 The results show the effect of DSS colitis on light / dye-induced thrombosis in the cremaster venules of mice;

[0042] Figure 13 The results show the effect of DSS colitis on light / dye-induced thrombosis in mouse cremaster arterioles;

[0043] Figure 14 The changes of TNF-α inflammatory factors in the colon of the experimental group mice were compared with those in the skeletal muscle;

[0044] Figure 15 The changes of IL-1β inflammatory factors in the colon of experimental group mice were compared with those in skeletal muscle;

[0045] Figure 16 The changes of IL-6 inflammatory factors in the colon of the experimental group mice were compared with those in the skeletal muscle;

[0046] Figure 17 The results of the lutein formula obtained in Examples 1-9 on the effect of thrombosis onset and stop time;

[0047] Figure 18 The level of TNF-α, a pro-inflammatory factor, in the colon of mice with colitis after intervention with the lutein composition obtained in Example 1-9;

[0048] Figure 19 The level of pro-inflammatory factor IL-1β in the colon of mice with colitis after intervention with the lutein composition obtained in Example 1-9;

[0049] Figure 20 The inflammatory factor IL-6 level in the colon of colitis mice after intervention with the lutein composition obtained in Example 1-9;

[0050] Figure 21 The results of arterial thrombus flow time in mice after intervention of colitis mice with the lutein compositions obtained in Examples 2, 4 and 7;

[0051] Figure 22 The results of the neutrophil count in mice after the lutein compositions obtained in Examples 2, 4 and 7 were used to intervene in colitis mice;

[0052] Figure 23 The thrombus weight results of mice after intervention with the lutein compositions obtained in Examples 2, 4 and 7 on colitis mice;

[0053] Figure 24 The thrombus length results of mice after intervention with the lutein compositions obtained in Examples 2, 4 and 7 in colitis mice;

[0054] Figure 25 The results of tail bleeding time of mice after intervention of colitis mice with the lutein compositions obtained in Examples 2, 4 and 7;

[0055] Figure 26 The level of TNF-α, a pro-inflammatory factor, in the colon of mice with colitis after intervention with the lutein compositions obtained in Examples 2 and 4;

[0056] Figure 27 The level of pro-inflammatory factor IL-1β in the colon of mice with colitis after intervention with the lutein compositions obtained in Examples 2 and 4;

[0057] Figure 28 The inflammatory factor IL-6 level in the colon of colitis mice after intervention with the lutein compositions obtained in Examples 2 and 4;

[0058] Figure 29 The thrombus weight results of mice after intervention with the lutein compositions obtained in Examples 2 and 4 on colitis mice;

[0059] Figure 30 The results of arterial thrombus flow time in mice after intervention of colitis mice with the lutein compositions obtained in Examples 2 and 4;

[0060] Figure 31 The effect of the lutein composition obtained in Examples 2 and 4 on the expression level of EPCR in mouse microvascular endothelial cells;

[0061] Figure 32 The effect of the lutein compositions obtained in Examples 2 and 4 on the expression level of TM in mouse microvascular endothelial cells;

[0062] Figure 33 The effect of the lutein composition obtained in Examples 2 and 4 on the expression level of EPCR mRNA in mouse microvascular endothelial cells;

[0063] Figure 34 The effects of the lutein compositions obtained in Examples 2 and 4 on the expression level of TM mRNA in mouse microvascular endothelial cells are shown. DETAILED DESCRIPTION

[0064] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0065] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0066] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0067] Among them, marigold flower granules were purchased from the marigold flower planting base in Xihe County, Longnan City, Gansu Province.

[0068] The boiling points of the components in liquefied butane and propane are mostly below 0°C, among which the boiling point of propane is -42.07°C and the boiling point of butane is -0.5°C. They are gases at room temperature and pressure of 37°C and become liquid after pressurization.

[0069] Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±3°C.

[0070] Example 1

[0071] 1. Lutein extraction and preparation

[0072] (1) Pretreatment of marigold particles: The marigold particles are first sent to the pretreatment workshop and pretreated in the following processes: screening, magnetic separation, stone removal, classification, etc. After removing the sand, gravel, broken particles and metal debris, they are used in the leaching workshop.

[0073] (2) Leaching: Using liquefied butane and propane (the volume ratio of liquefied butane to liquefied propane is 1:1.28) as the extraction solvent, the treated marigold flower particles are transported to the extractor through a scraper conveyor, and mixed with the extraction solvent at a material-liquid mass ratio of 1:2.5. The solvent is continuously leached through 6 solvent pumps, wherein the leaching temperature is 40 ° C, the pressure is 0.87 MPa, and the leaching time is 56 minutes. Lutein is continuously countercurrently leached by the extraction solvent and dissolved into one. The appearance of the solution is clear, and after adding lutein, the solution becomes turbid, indicating that the lutein has exceeded the solubility of the solvent at the current temperature, and the solution is in a supersaturated state, forming a saturated solution; after leaching, it is filtered through a 150-mesh net in the silo to separate the filter residue from the extract;

[0074] (3) The obtained filter residue is evaporated by a residue evaporator at 124°C with negative pressure steam. The residue evaporator evaporates the extraction solvent in the filter residue and enters a condenser for cooling and recycling. The residue is transported to the residue storage bin through an auger.

[0075] (4) Evaporation and concentration: The obtained extract is pumped into a cyclone for separation, and then the obtained saturated solution is added to an evaporator for evaporation, wherein the vacuum pressure in the evaporator is 0.048 MPa. Under the separation of the flash box in the evaporator, the vapor phase solvent obtained by evaporation enters a refrigerated separator for cooling, recovery and recycling; the liquid phase enters another evaporator for secondary evaporation, and again under the separation of the flash box, the vapor phase solvent obtained by secondary evaporation enters a refrigerated separator for cooling, recovery and recycling; the liquid phase enters a distillation tower with a vacuum degree of 0.048 MPa and is distilled and concentrated at a temperature of 60°C to obtain lutein extract.

[0076] (5) Removal of disability

[0077] The evaporated extract is further treated to remove residual solvent, and the concentrated extract is treated with pigments using a high-speed centrifuge to further reduce the insoluble matter content, thereby obtaining lutein.

[0078] A lutein composition comprising the following raw materials in different weight fractions:

[0079] Lutein 8%, zeaxanthin 5%, curcumin 4%, quercetin 3%, fucoidan 10%, honeysuckle extract 5%, casein 1%, beeswax 1%, soy lecithin 1%, soy oil 29%, glycerin 23% and hydroxypropyl methylcellulose 10%.

[0080] A method for preparing a lutein composition (soft capsule) comprises the following steps:

[0081] 1. Capsule shell preparation (gelatinization): gelatin: glycerin: water = 1:0.35:0.8. Add glycerin and water into the gelatinization tank, heat to 68℃±2℃, then add gelatin. Sol for about 1 hour, vacuum to remove bubbles and moisture, so that the gelatin is completely dissolved without particles. The particle size index can reach 15,000 to 25,000 cp, thus meeting the product requirements.

[0082] 2. Preparation of molten suspending and dispersing agent R1: Mix soybean oil, beeswax and soybean lecithin and heat them in a water bath to 70℃±3℃ to melt to obtain R1 melt.

[0083] 3. Content preparation (ingredients): Weigh the raw materials of the lutein composition described above, crush and mix them evenly, then mix them evenly with the R1 melt, stir them in a high-shear dispersing emulsifier for 2 minutes, and heat to 90-95°C and grind and mix them evenly to obtain the capsule contents for later use.

[0084] 4. Pill pressing: Add the contents into the hopper of the pill press, control the spray temperature within the range of 38-45°C, the glue temperature within the range of 55-70°C, the cooling drum temperature within the range of 15-26°C, the liquid temperature not higher than 42°C, and press the pills into shape using the pill press.

[0085] 5. Drying: Place the freshly pressed soft capsules in a rotating cage and air dry them dynamically during the rotation. The setting time is about 2 to 4 hours. Reduce the moisture content to between 25 and 30%, and the soft capsule shell becomes elastic and does not deform.

[0086] Example 2

[0087] A method for preparing lutein is the same as that in Example 1.

[0088] A lutein composition comprising the following raw materials in different weight fractions:

[0089] Lutein 8%, zeaxanthin 12%, curcumin 8%, quercetin 3%, fucoidan 13%, honeysuckle extract 8%, casein 0.8%, beeswax 1.2%, soy lecithin 1.8%, soy oil 20.2%, glycerin 19% and hydroxypropyl methylcellulose 5%.

[0090] A method for preparing a lutein composition is the same as that in Example 1.

[0091] Example 3

[0092] A method for preparing lutein is the same as that in Example 1.

[0093] A lutein composition comprising the following raw materials in different weight fractions:

[0094] Lutein 0, Zeaxanthin 21%, Curcumin 19%, Quercetin 5%, Fucoidan 14%, Honeysuckle Extract 11.3%, Casein 1.5%, Beeswax 0.8%, Soybean Lecithin 0.4%, Soybean Oil 8.5%, Glycerin 11% and Hydroxypropyl Methylcellulose 7.5%.

[0095] A method for preparing a lutein composition is the same as that in Example 1.

[0096] Example 4

[0097] A method for preparing lutein is the same as that in Example 1.

[0098] A lutein composition comprising the following raw materials in different weight fractions:

[0099] Lutein 10.5%, zeaxanthin 5%, curcumin 8%, quercetin 9%, fucoidan 10%, honeysuckle extract 9.8%, casein 0.8%, beeswax 0.6%, soy lecithin 0.6%, soy oil 24.2%, glycerin 14% and hydroxypropyl methylcellulose 7.5%.

[0100] A method for preparing a lutein composition is the same as that in Example 1.

[0101] Example 5

[0102] A method for preparing lutein is the same as that in Example 1.

[0103] A lutein composition comprising the following raw materials in different weight fractions:

[0104] Lutein 10.5%, Zeaxanthin 12%, Curcumin 16%, Quercetin 9%, Fucoidan 13%, Honeysuckle Extract 11%, Casein 1.5%, Beeswax 0.8%, Soybean Lecithin 0.7%, Soybean Oil 7.5%, Glycerin 11% and Hydroxypropyl Methylcellulose 7%.

[0105] A method for preparing a lutein composition is the same as that in Example 1.

[0106] Example 6

[0107] A method for preparing lutein is the same as that in Example 1.

[0108] A lutein composition comprising the following raw materials in different weight fractions:

[0109] Lutein 0%, zeaxanthin 21%, curcumin 6.5%, quercetin 14%, fucoidan 14%, honeysuckle extract 6%, casein 1%, beeswax 0.5%, soy lecithin 0.5%, soy oil 20%, glycerin 11% and hydroxypropyl methylcellulose 5.5%.

[0110] A method for preparing a lutein composition is the same as that in Example 1.

[0111] Example 7

[0112] A method for preparing lutein is the same as that in Example 1.

[0113] A lutein composition comprising the following raw materials in different weight fractions:

[0114] Lutein 13.5%, zeaxanthin 5%, curcumin 16%, quercetin 21%, fucoidan 10%, honeysuckle extract 11.3%, casein 1.5%, beeswax 0.8%, soy lecithin 0.4%, soy oil 5.5%, glycerin 8% and hydroxypropyl methylcellulose 7%.

[0115] A method for preparing a lutein composition is the same as that in Example 1.

[0116] Example 8

[0117] A method for preparing lutein is the same as that in Example 1.

[0118] A lutein composition comprising the following raw materials in different weight fractions:

[0119] Lutein 13.5%, zeaxanthin 12%, curcumin 4%, quercetin 21%, fucoidan 13%, honeysuckle extract 6%, casein 1%, beeswax 0.4%, soy lecithin 0.6%, soy oil 9%, glycerin 14% and hydroxypropyl methylcellulose 5.5%.

[0120] A method for preparing a lutein composition is the same as that in Example 1.

[0121] Example 9

[0122] A method for preparing lutein is the same as that in Example 1.

[0123] A lutein composition comprising the following raw materials in different weight fractions:

[0124] Lutein 17.5%, zeaxanthin 0%, curcumin 11.5%, quercetin 21%, fucoidan 14%, honeysuckle extract 20%, casein 0.8%, beeswax 0.9%, soy lecithin 0.6%, soy oil 4.2%, glycerin 6% and hydroxypropyl methylcellulose 3.5%.

[0125] A method for preparing a lutein composition is the same as that in Example 1.

[0126] Technical effects:

[0127] Animal model construction

[0128] (1) Materials and methods

[0129] Male C57BL / 6 mice (Jackson Laboratories, ME) were used as experimental animals. All mice were housed in standard cages under specific pathogen-free conditions and fed standard laboratory chow and water until the desired age (6-8 weeks). All animal procedures were reviewed and approved by the Animal Care and Use Committee, and experimental animals were managed in strict accordance with the AAALAC guidelines and the 3R principles of animal welfare.

[0130] DSS-induced colitis: Mice in the experimental group were fed a solution of 3% (wt / vol) DSS (mol wt, 40,000) dissolved in filtered drinking water. The first day of DSS feeding was defined as day 0, and mice were maintained on DSS feeding until day 6.

[0131] The mice in the control group were given normal saline (without DSS).

[0132] Assessment of Colitis Progression: While mice were receiving DSS, body weight, stool status, the presence of occult blood in the stool, and perianal bleeding were observed and recorded daily. The Disease Activity Index (DAI), a measure of disease severity from 0 to 4, was calculated based on collected data on stool consistency, the presence of blood in the stool, and weight loss. The DAI was monitored to ensure that the clinical response induced by DSS was consistent with colitis disease activity.

[0133] Cremaster muscle preparation: On day 6 of DSS (experimental group) or water (control) colitis induction, mice were anesthetized intraperitoneally with 50 mg / kg sodium pentobarbital and given supplementary doses (12.5 mg / kg) as needed. The right internal jugular vein was cannulated for intravenous administration of FITC-dextran. Throughout the experiment, body temperature was maintained at 35.5-36.5°C using a homeothermic blanket and monitored with a rectal temperature probe. The cremaster muscle was prepared for intravital fluorescence microscopy as described above. The exposed cremaster muscle surface was continuously filled with bicarbonate-buffered saline with a pH of 7.35-7.45. The cremaster muscle preparation was moved to the stage of an upright fluorescence microscope and stabilized for 20-30 minutes before the start of the experiment.

[0134] An upright microscope (BX51WI; Olympus, Tokyo, Japan) was used, equipped with a 40× water immersion objective (LUMPlanFI / IR40× / 0.80W). Microscopic images were projected onto a monitor (SonyTRINITRON PVM-2030) using a color video camera (Hitachi VK-C150) and recorded using a DVD recorder (JVC SR-MV50). A video timer (Panasonic Time-Date Generator WJ-810) was connected to the monitor to record time and date. The diameter of the cremaster vessels was measured on a personal computer (G4 Macintosh, Apple) using analysis software (ImageJ 1.37v, NIH, Public Domain software). After the preparation was stabilized, 10 ml / kg of 5% FITC-dextran (150,000 MW; Sigma, St. Louis, MO) was slowly injected intravenously. It was allowed to circulate for 10 min, and then a diameter of 30-50 μm was selected for study.

[0135] (2) Experimental plan

[0136] Grouping and treatment of mice in the light / dye endothelial injury model:

[0137] 1) Control group mice (no DSS in drinking water), 2) Experimental group DSS-treated (colitis) mice, 3) Experimental group mice were gavage-fed with the lutein composition obtained in Example 1-9 5 minutes before vascular epi-irradiation, at a feeding amount of 5 mg / kg.

[0138] Wherein, the gavage feeding method comprises the following steps:

[0139] Dilute the lutein softgel solution 100-fold (with saline) and administer to the mouse via gavage at a concentration of 5 mg / kg body weight. Aim the gavage needle vertically at the mouse's mouth and insert it vertically, along the corner of the mouth, against the mouse's upper jaw. Secure the needle firmly and do not shake it. Slowly inject the solution into the mouse's stomach. Remove the needle and return the mouse to its original cage. Clean the needle and save it for future use.

[0140] Light / dye-induced thrombosis:

[0141] Thrombosis was studied in randomly selected second- or third-order venules and arterioles (1-3 per mouse) within each cremaster muscle that were characterized by a diameter of 35-50 μm, a length of at least 100 μm, and a wall shear rate of ≥500 / s. Then, 10 ml / kg of 5% FITC-dextran (150,000 MW) (Sigma chemicals, St. Louis, MO) was slowly infused into the intravenous cannula and allowed to circulate for 10 minutes. Photoactivation of FITC-dextran (excitation: 495 nm, emission: 519 nm) within the microvasculature was achieved by epi-illumination using a 175 W xenon lamp (Lambda LS, Sutter, CA) and a fluorescein filter cube (HQ-FITC, Chroma Technology Company, VT). Excitation power density was measured daily (ILT 1700 radiometer, SED033 detector, International Light, MA) and maintained within 1% of 0.74 W / cm. Epi-illumination was applied continuously to the blood vessels, and thrombus formation was quantified by determining: 1) the onset of platelet deposition / aggregation within the microvessels (onset time), and 2) the time required to completely stop blood flow for ≥60 seconds (stop time). Epi-illumination was stopped once blood flow in the study vessel ceased. Results for each vessel type (venules, arterioles) were averaged from 2-4 thrombi generated per mouse.

[0142] Mesenteric artery thrombosis modeling steps:

[0143] FeCl3 induced thrombosis in mouse mesenteric arterioles by in vivo microscopy: After anesthetizing mice with 1.5% isoflurane (RWD), rhodamine 6G (Sigma, 83697) was injected into the mouse bloodstream via the tail vein for 10 minutes to label platelets and leukocytes. A 1×1 mm filter paper soaked in 5% FeCl3 (MACKLIN, I809489) was used to treat superior mesenteric arteries with a diameter of ≈100 μm. Five minutes after injury, the filter paper was removed, and residual FeCl3 was washed away with PBS. Vascular occlusion was monitored and retimed for 15 minutes under a Leica inverted microscope (CTR7000 HS), and the mice were kept at constant temperature throughout the experiment. Only one arteriole was selected for each mouse.

[0144] Inferior vena cava (IVC) stenosis:

[0145] Inferior vena cava stenosis was used to induce deep venous thrombosis. After mice were anesthetized with isoflurane, the skin and peritoneum were incised along the midline of the abdomen to expose the abdominal cavity. The intestines were removed and wrapped with gauze soaked in 37°C saline. All branches of the IVC were ligated, and the IVC was carefully separated from the aorta below the angle between the left renal vein and the IVC. The inferior vena cava was then ligated with polypropylene suture and a 30-gauge needle and then removed to avoid endothelial detachment. The peritoneum and skin were sutured continuously, and the thrombus was removed 48 hours later for length and body weight measurement.

[0146] Tail bleeding assay:

[0147] Isoflurane-anesthetized mice were placed on a heating plate to maintain body temperature, 5 mm was cut from the tip of the tail to cause a coherent injury, and the tail was immediately immersed in 15 mL of 37 ° C normal saline, and the bleeding time was recorded until the bleeding stopped or until 10 minutes. Hemostasis > 1 min was considered to be the end of bleeding, and the tail was removed in time. The hemoglobin content was measured using a spectrophotometer to indicate the amount of blood loss of the mouse. After removing the supernatant, 900 μL of water was added to resuspend and lyse the red blood cells. After 17 s, 100 μL of PBS was added to restore the osmotic pressure, and the cells and debris were removed by centrifugation (10000 rpm, 5 min), and the supernatant containing hemoglobin was retained for 550 nm optical density detection (OD550).

[0148] 2. Confirm the success of DSS modeling

[0149] Clinical and Macroscopic Analysis of Murine Colitis:

[0150] Mice were monitored daily for clinical analysis of colitis, including body weight, diarrhea, and bleeding. Weight loss was calculated as the percentage difference relative to initial body weight. Mice were euthanized on day 7 after DSS administration. The colon was resected and opened longitudinally. Macroscopic lesions were measured by a blinded observer using the following scoring system: 0: normal; 1: congestion, edema, no ulcers; 2: congestion, edema, small linear ulcers or petechiae; 3: congestion, edema, wide ulcers, necrosis or adhesions; 4: congestion, edema, megacolon, stricture or perforation.

[0151] The results of DSS-induced colitis mice in terms of weight loss, diarrhea, bloody stools and macroscopic scores were as follows: Figure 1-4 As shown. Figure 1 It can be seen that the weight of the mice in the experimental group decreased significantly compared with the control group. Figure 2 and Figure 3 It can be seen that the diarrhea and bloody stool scores of the experimental group mice were higher than those of the control group. Figure 4 It can be seen that the macroscopic scores of the mice in the experimental group were significantly increased compared with those in the control group. These results indicate that the DSS-induced colitis mouse model in the experimental group was successfully established.

[0152] 3. Experimental studies confirming that IBD mice are more susceptible to thrombosis

[0153] The superior mesenteric artery, which has a diameter of approximately 100 μm, was used to study whether IBD mice are more susceptible to thrombosis. To this end, a rhodamine 6G solution was injected into the tail vein and white blood cells and platelets in the peripheral blood of mice were labeled. The mesenteric arterioles damaged by FeCl3 were monitored by intravital microscopy, and the time to complete blood flow occlusion was recorded. In addition, thrombosis was observed using the inferior vena cava stenosis model (a widely used model for inducing deep vein thrombosis in mice), and thrombi were removed for measurement 48 hours after surgery. The results are shown in Figure 2. Figures 5 to 11 shown.

[0154] from Figure 5 It can be seen that the vascular occlusion time of the experimental group mice was shorter than that of the control mice, and there was a significant difference between the two groups (P < 0.01). In addition, peripheral blood cell analysis was performed, and the test results ( Figure 6 and Figure 7 ) It can be seen that the neutrophil count of the experimental group mice increased significantly compared with the normal control group mice (P < 0.01), while there was no statistical difference in the platelet count between the two groups (P > 0.05). Figure 8 It can be seen that the thrombus weight of the mice in the experimental group increased significantly compared with the control group, and the difference between the two groups was significant (P < 0.01). Figure 9It can be seen that compared with the control group mice, the thrombus length of the experimental group mice was significantly increased, and there was a certain difference between the two (P < 0.05). In addition, the tail bleeding time and hemoglobin level of the experimental group mice were significantly reduced compared with the normal group mice (P < 0.01) ( Figure 10 and Figure 11 ). In summary, it can be seen that the DSS-induced colitis mice in the experimental group are more likely to develop thrombosis than the healthy mice in the control group.

[0155] 4. Effects of DSS colitis on light / dye-induced thrombosis in the arterioles and venules of the cremaster muscle in mice Figure 12 and Figure 13 As shown, the time required for thrombosis to initiate and complete stasis was significantly longer in arterioles than in venules. Although there were no differences in onset or stasis between venules from control and experimental mice, both variables were significantly reduced in the cremaster arterioles of experimental mice compared with control mice. Because DSS-induced enhanced extraintestinal thrombosis was not observed in mouse veins, all subsequent experiments focused on thrombosis studies in arterioles.

[0156] 5. Study on the effect of lutein formula on changes in inflammatory factors

[0157] Proinflammatory cytokines are considered to be an important link between inflammation and the hypercoagulable and prothrombotic state observed in certain pathological conditions. Therefore, we examined the changes in several inflammatory factors (TNF-α / IL-6 / IL-1β) in the colon and skeletal muscle of mice with colitis and thrombosis. Figure 14-16 shown.

[0158] from Figure 14-16 The results showed that the changes in TNF-α, IL-1β, and IL-6 in the colon of the experimental group mice were more obvious than those in skeletal muscle. Therefore, the changes in inflammatory factors in the colon were selected for subsequent studies.

[0159] 6. The effect of lutein combination on thrombosis in cremaster arterioles was investigated. Figure 17 shown.

[0160] from Figure 17It can be seen that the lutein composition obtained in Examples 1-9 (Y1-Y9) has a certain effect on both the start time and the stop time of thrombosis, but the effect on the stop time is more obvious. In addition, it can be seen from the figure that the extraintestinal thrombosis stop time of the DSS model group mice is significantly shorter than that of the control group mice, and there is a significant difference between the two (P < 0.01), indicating that the experimental group mice have obvious extraintestinal thrombosis. When the lutein composition obtained in Examples 1-9 was used to intervene in the treatment of colitis mice, the extraintestinal thrombosis phenomenon of the colitis mice was alleviated to varying degrees. Figure 17 We can further find that after using the lutein composition obtained in Example 4 to intervene in mice with colitis and extraintestinal thrombosis, the improvement effect of extraintestinal thrombosis is the most obvious. The time for extraintestinal thrombosis to stop flowing is significantly increased compared with the model group mice (DSS group), and there is a significant difference between the two (P < 0.01); secondly, the lutein composition obtained in Example 2 also has a good effect on improving extraintestinal thrombosis in mice. After intervention with the lutein composition obtained in Example 2, it can be seen that the time for extraintestinal thrombosis to stop flowing in mice with colitis is significantly increased compared with the model group mice (DSS group) (P < 0.01). Again, from Figure 17 As can be seen, the lutein composition obtained in Example 7, after intervention on mice with colitis and intestinal thrombosis, had a poorer effect on improving intestinal thrombosis than did Examples 2 and 4, but still showed a difference compared to the model group (DSS group) mice (P < 0.05), indicating that the lutein composition obtained in Example 7 also had a certain improvement effect on the symptoms of mice with colitis and intestinal thrombosis. At the same time, the lutein composition obtained in Example 5 also had a similar effect as Example 7. In addition, it can be found that the other lutein formulas (Examples 1, 3, 6, 8, and 9) had almost the same improvement effect on intestinal thrombosis in mice with colitis, but the improvement effect was far less than that of Examples 2, 4, and 7, and there was no difference compared to the model group (DSS group). However, Examples 3, 6, and 9 showed a certain difference compared to the normal control group mice (P < 0.05). This change indicates that the lutein compositions obtained in Examples 3, 6, and 9 had little effect on improving the symptoms of intestinal thrombosis in mice with colitis. Therefore, in subsequent studies, it is not necessary to continue studying these three formulas.

[0161] 7. Effects of lutein formula on changes in three inflammatory factors in the colon of colitis mice

[0162] To further verify the conclusions, and confirm the effect of the lutein compositions obtained in Examples 1, 3, 6, and 9 on improving inflammation and extraintestinal thrombosis in colitis mice, we further conducted experiments on the effects of the 9 lutein formulations on the changes in inflammatory factors in colitis mice to further confirm whether the lutein formulations of the present invention regulate colitis-related thrombosis by affecting the changes in these three inflammatory factors in the colon. Figure 18-20 shown.

[0163] 1) From Figure 18 As can be seen, after the lutein compositions obtained in Examples 2, 4, and 7 were used to intervene in colitis mice, the level of the pro-inflammatory factor TNF-α in the colon of the mice decreased significantly, showing a significant difference compared to the model group (DSS group) mice (P < 0.01). Of course, there were also some differences between the mice in Examples 1, 5, and 8 and the model group (DSS group) (P < 0.05), but the intervention effects on colitis mice were still slightly inferior to those of Examples 2, 4, and 7. In contrast, the inflammation-improving effects of Examples 3, 6, and 9 on colitis mice were relatively weak, and their effects on the inflammatory factor TNF-α were almost similar to those of the model group (DSS group), indicating that the intervention effects of Examples 3, 6, and 9 on inflammatory factors were not ideal.

[0164] 2) From Figure 19 It can be seen from the improvement effects of each formula on the proinflammatory factor IL-1β that the intervention effect of the lutein composition obtained in Examples 2, 4 and 7 is still the most ideal, and there is a significant difference compared with the model group (P < 0.01). The intervention effects of Examples 5 and 8 are second best, and there is a certain difference compared with the model group (P < 0.05). However, the improvement effects of Examples 3, 6 and 9 on the proinflammatory factor IL-1β in the colon of colitis mice are relatively poor, and the improvement effect is almost 0. The change of IL-1β is similar to that of the model group. This also shows that the lutein composition obtained in Formula Examples 3, 6 and 9 has a very poor effect on improving inflammation in colitis mice.

[0165] 3) From Figure 20 The intervention effects of each formula on the inflammatory factor IL-6 can be seen to have a similar trend to the intervention effects of the previous two inflammatory factors, that is, Example 2 and Example 4 have the most ideal improvement effects on IL-6, although their improvement effects are slightly inferior to those of the previous two factors. This may be because in the colon of colitis mice, the degree to which the inflammatory factor IL-6 participates in the inflammatory process or the corresponding signal pathway is weaker than that of the previous two inflammatory factors. Therefore, the degree of change in its detection process is weaker. However, even if the degree of IL-6 participation in inflammation is weaker, Figure 13 It can still be seen from the results that the lutein compositions obtained in Examples 3, 6 and 9 had the worst intervention effects, and the effects after intervention were almost not improved compared with the model group, indicating that the prescription effects of these three groups were the worst.

[0166] 4) Therefore, through the changes in the three inflammatory factors, we further verified our conclusion that the lutein compositions obtained in Examples 3, 6, and 9 had the worst effect on improving colon inflammation and the accompanying extraintestinal thrombosis in colitis mice, and we will not explore these three compositions in the future.

[0167] 8. Effects of the three best prescriptions on mesenteric artery thrombosis

[0168] The three lutein compositions with better intervention effects (lutein compositions obtained in Examples 2, 4 and 7) were tested again to explore their effects on mesenteric artery thrombosis. The arterial thrombus flow time, thrombus weight, thrombus length, neutrophil count and tail bleeding time were tested respectively. The test results are as follows Figures 21 to 25 .

[0169] 1) From Figure 21 It can be seen that the arterial thrombosis flow time of the DSS colitis mice was significantly reduced compared with the model group mice (P < 0.01), while the arterial thrombosis flow time of the mice intervened by the three lutein formulas increased to varying degrees. Among them, the intervention effect of Example 4 was the most ideal. The arterial thrombosis flow time after intervention was significantly increased compared with the model group mice (DSS group), and there was a significant difference between the two (P < 0.01). Of course, the intervention effect of Example 2 was also good. The arterial thrombosis flow time after intervention rebounded to a certain extent compared with the model group, and there was a certain difference between the two (P < 0.05). However, the improvement effect of Example 7 on the change of arterial thrombosis flow time after intervention was not obvious, and there was no difference compared with the model group.

[0170] 2) From Figure 22 As can be seen, the number of neutrophils in mice with DSS colitis showed a significant increase compared to the normal group. However, after intervention with the three lutein formulas, the change in neutrophil count decreased to a certain extent compared to the model group (DSS group), but there was no significant difference. The differences between the three formulas were not significant. This may be because the effects of the three lutein formulas on neutrophil count were not very different. We can continue to study other indicators to distinguish the differences in the intervention effects of the three formulas.

[0171] 3) From Figure 23 It can be seen that compared with the model group mice, the thrombus weight was significantly reduced after intervention with the lutein formula, among which the intervention effects of Example 2 and Example 4 were the most ideal, and there were significant differences between them compared with the model group mice (P < 0.01). Of course, the intervention effect of Example 7 was also good, and it showed certain differences compared with the model group (P < 0.05), but compared with Example 2 and Example 4, its intervention effect was second.

[0172] 4) From Figure 24 It can be seen that in terms of the effect on thrombus length, after Example 4 intervened in colitis mice, the degree of reduction in thrombus length was significantly different from that of the model group (P < 0.01); the degree of reduction in thrombus length after intervention in Example 2 and Example 7 was also different from that of the model group (P < 0.05), but the improvement effect was not as good as that of Example 4.

[0173] 5) From Figure 25 It can be seen that Example 4 is still better in terms of the effect on blood flow time. Compared with the model group, the blood flow time of the mice in the Example 4 group increased significantly, and there was a significant difference between the two groups (P < 0.01). After intervening in the colitis mice, although the blood flow time of Example 2 and Example 7 increased to a certain extent compared with the model group, the difference was not significant (P < 0.05), indicating that the improvement effect of Example 2 and Example 7 on the symptoms of enteritis with thrombosis is still weaker than that of Example 4.

[0174] From the above examples, it can be seen that whether it is the changes in the start and stop time of thrombosis in the cremaster arterioles or the changes in the three inflammatory factors, a consistent pattern can be seen, that is, three lutein compositions (Examples 2, 4, and 7) have a more ideal intervention effect on extraintestinal thrombosis and colon inflammation. Further comparison shows that among the three better formulas (Examples 2, 4, and 7), Examples 2 and 4 have a more significant improvement effect on intestinal inflammation and thrombosis than Example 7. Therefore, the effects of the two best formulas (Examples 2 and 4) on intestinal inflammation and corresponding thrombosis will be further studied.

[0175] 9. The effects of the lutein compositions obtained in Examples 2 and 4 on arterial thrombosis and changes in inflammatory factors were investigated. The results are as follows: Figure 26-30 shown.

[0176] (1) From Figure 26 It can be seen that the pro-inflammatory factor TNF-α in the colon of DSS colitis mice was significantly increased compared with the normal group mice (P < 0.01), while after the intervention of Example 4, TNF-α was significantly reduced (P < 0.01); the pro-inflammatory factor TNF-α in the colitis mice intervened by Example 2 also decreased to a certain extent, which was different from the model group (P < 0.05), but the improvement effect was not as obvious as that of Example 4.

[0177] (2) From Figure 27 It can be seen that Examples 2 and 4 have comparable intervention effects on the inflammatory factor IL-1β in colitis mice, and the effects of the two groups after intervention are significantly different from those in the model group of colitis mice (P < 0.01), indicating that Examples 2 and 4 are equivalent in improving the inflammatory factor IL-1β.

[0178] (3) From Figure 28It can be seen that in terms of the intervention effect of IL-6, although Example 2 showed some improvement compared with the model group, there was no difference (ns), and after intervention in Example 4, the inflammatory factor IL-6 decreased to a certain extent compared with the colitis mice in the model group, but the difference between the two groups was not obvious (P < 0.05). This may be because the change of IL-6, an inflammatory factor, in the symptoms of colitis with thrombosis is not obvious, that is, IL-6 may not be a more obvious marker in the relevant signal system of changes in colitis with thrombosis symptoms, resulting in the response of Examples 2 and 4 to this inflammatory factor being relatively weak compared with other inflammatory factors (TNF-α, Il-β).

[0179] (4) From Figure 29 It can be seen that in terms of the effect on thrombus length, the thrombus length of the colitis mice after intervention in Example 4 was significantly reduced, with a significant difference compared with the model group (P < 0.01); the thrombus length after intervention in Example 2 was reduced to a certain extent compared with the model group, but the difference was not significant (P < 0.05).

[0180] (5) From Figure 30 It can be seen that for the effect on the onset time of thrombosis, the effect of each formula after intervention did not change significantly, which is consistent with the results of the previous study, because the lutein formula needs a certain time to intervene in inflammation and thrombosis, and the onset time of thrombosis may be relatively short, and the response change is not obvious; and for the effect on the cessation time of thrombosis, both formulas showed relatively ideal intervention effects, but it can be seen that the intervention effect of Example 4 is still the most ideal, and its effect after intervention is significantly different from that of the model group (P < 0.01), while the effect of Example 2 after intervention is different from that of the model group but not significant (P < 0.05).

[0181] In summary, the lutein composition obtained by the present invention can effectively improve the symptoms of colitis with extraintestinal thrombosis. Animal experiments have confirmed that Example 4 has the most ideal intervention effect on colitis with extraintestinal thrombosis, followed by Example 2. The present invention can effectively improve and restore the symptoms of colitis with extraintestinal thrombosis by improving the changes in inflammatory factors TNF-α, IL-1β, and IL-6, the changes in neutrophil count related to the inflammation-coagulation pathway, and the length and weight of thrombi.

[0182] 10. Preliminary study of pharmacological mechanisms

[0183] Through the above experiments, it has been confirmed that Examples 2 and 4 are the two most ideal formulas for intervening in the symptoms of colitis with intestinal thrombosis. Further research was conducted on the possible mechanisms of action of these two lutein formulas in improving the symptoms of colitis with intestinal thrombosis. Two key cytokines of the intestinal inflammation-related microvascular system - thrombomodulin (TM) and endothelial PC receptors were further detected by flow cytometry to study whether the lutein composition can improve colitis with intestinal thrombosis through PC dredging.

[0184] Experimental steps:

[0185] To determine the expression of TM and EPCR in HIMECs, endothelial cells were seeded at a density of 5×10 on fibronectin-coated wells of a 24-well cluster plate (4 cells / ml per well). The confluent monolayer of cells was cultured in culture medium for 24 hours. At the end of the culture period, HIMECs were washed five times in cold PBS, and single-cell suspensions were obtained using separation buffer (PBS; 20 mM HEPES, pH 7.4; 10 mM EDTA; and 0.5% BSA), followed by vigorous pipetting. After additional washing, HIMECs were incubated with first-generation anti-TM or anti-EPCR; the above HIMEC stimulation did not alter cell viability as assessed by trypan blue staining (data not shown). Samples were analyzed by quantitative flow cytometry using a Coulter Epics XL flow cytometer (Beckman Coulter). The experimental results are shown in Figures 31-34 :

[0186] from Figures 31-34 It can be seen that the lutein composition obtained by the present invention affects the expression levels of EPCR and TM in microvascular endothelial cells, especially the mRNA levels. Both the EPCR expression level and the TM expression level increased significantly after intervention with the lutein composition compared to the colitis mice (DSS group), and there were differences between the groups (P < 0.01, P < 0.05). Because EPCR and TM are two important protein molecules in the PC signaling pathway, it is predicted that the lutein composition obtained by the present invention can achieve an ideal intervention effect in improving colitis with extraintestinal thrombosis through PC dredging.

[0187] The present invention only conducts a preliminary and superficial exploration of the mechanism of the lutein composition in intervening in the symptoms of colitis with extraintestinal thrombosis, and further verification is needed in the future through more animal experiments and clinical trials.

[0188] The present invention obtains natural lutein by low-temperature extraction of marigold flowers under specific temperature, pressure and solvent conditions, and further obtains multiple lutein formulas. Animal experimental studies have confirmed that the lutein compositions obtained in Examples 2 and 4 of the present invention have a relatively ideal improvement effect on the symptoms of colitis with extraintestinal thrombosis. Its mechanism of action may involve one or more signal pathways related to the inflammation-coagulation system. It can regulate the expression levels of inflammatory factors and related proteins or receptors on the related signal pathways, which can provide reference significance for the future clinical development of drugs for the treatment of diseases such as colitis and thrombosis or atherosclerosis. In addition, the lutein in the lutein formula of the present invention is extracted from marigold flower granules. The raw materials are natural, and the other raw materials are all medicinal and edible ingredients. There are no side effects. It can prevent and improve inflammatory bowel disease and symptoms such as thrombosis. It is of great significance, especially for middle-aged and elderly people who are prone to suffering from these two diseases at the same time. It can greatly reduce the treatment pain of the middle-aged and elderly patients, improve patient compliance, and thus improve the quality of life of the middle-aged and elderly people.

[0189] The above are merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A lutein composition, characterized in that Including the following raw materials by mass fraction: Lutein 8-13.5%, zeaxanthin 5-12%, curcumin 8-16%, quercetin 3-21%, fucoidan 10-13%, honeysuckle extract 6-11.3%, casein 0.8-1.5%, beeswax 0.4-1.2%, soybean lecithin 0.4-1%, soybean oil 5.5-24.2%, glycerin 8-19% and hydroxypropyl methylcellulose 5-7.5%; The preparation method of lutein comprises the following steps: The marigold flower particles are extracted using propane and butane as extraction solvents, and the obtained extract is evaporated twice and then distilled and concentrated, and the obtained extract is then centrifuged after removing the solvent to obtain the lutein; In the extraction solvent, the volume ratio of propane to butane is 1:(1.1-1.4); The extraction temperature is 40°C, the pressure is 0.8-0.9 MPa, and the time is 45-60 min; The evaporation temperature is 36°C ± 2°C and the pressure is 0.04-0.05MPa; The temperature of the distillation concentration is 60° C. and the pressure is 0.04-0.05 MPa.

2. A lutein composition according to claim 1, characterized in that: The mass ratio of the extraction solvent to the marigold flower particles is 1:(2-3).

3. A lutein composition according to claim 1 or 2, characterized in that: The dosage form of the lutein composition includes one or more of powder, emulsion, capsule and soft candy dosage forms.

4. Use of the lutein composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing and improving IBD and its thrombotic complications.

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